Recombinant nucleic acid technology has proven to be a powerful tool for the expression of the products encoded by nucleic acids of interest. This has resulted in the ability to produce polypeptides and nucleic acids for both research and commercial applications.
Some encoded products, however, are toxic to the cellular or host environment in which their expression occurs, either because the product is inherently toxic or because the levels at which expression occurs is so high as to result in toxicity. One means of dealing with this difficulty has been to use transient expression systems wherein the encoded product is expressed and recovered before toxicity results in reduced levels of product. Alternatively, the encoded product is placed under a tightly controlled regulation system such that the product may be expressed and then expression terminated before toxicity rises to lethal levels. One example of a tightly controlled regulatory system is seen with the use of a tetracycline regulated operator/promoter in combination with a tet repressor (see for example U.S. Pat. No. 5,750,396).
The expression of a toxic product is of particular importance in situations where the product must be continually expressed because it is a component of a larger product being produced, or metabolic activity being conducted, by the cell. One example of such a situation is in the case of a viral packaging cell line, which expresses products necessary for the assembly and packaging of viral particles. If any one of the necessary viral gene products is toxic to the cell, the need to control its expression becomes critical if a stable (as opposed to transient) packaging cell line is to be used. One example of a necessary toxic viral gene is in the case of the G protein from vesicular stomatitis virus (VSV), which is desirable for the production of pseudotyped viral particles.
An example using the tet operator and repressor to regulate the expression of VSV-G is described by Henriette et al. (J. Virol. 73(1):576-584, 1999), where the tet repressor (as a chimeric fusion product with a domain of VP-16 and referred to as tTA) is under the control of a cytomegalovirus (CMV) promoter and VSV-G is under the control of a tet operator. Expression of the chimeric repressor in the absence of tetracycline results in no expression of VSV-G. The presence of tetracycline prevents association between tTA and the tet responsive elements (TRE) found in the operator to allow the expression of VSV-G. This system is referred to as “tet-on” where the presence of tetracycline results in the expression of the gene of interest (i.e. VSV-G).
There is also an alternative “tet-off” system where tTA is a chimeric transactivator. It cannot bind to the TRE of a tet operator in the presence of tetracycline. But in the absence of tetracycline, tTA binds to the operator and strongly activates the promoter to express a coding sequence of interest.
Klages et al. (Molec. Therap. 2(2):170, 2000) teach the use of a similar two nucleic acid system to control VSV-G expression. The first nucleic acid expresses tTA which then controls a TRE containing tet operator that controls VSV-G expression. The same tTA protein also regulates expression of the rev protein which in turn regulates the expression of the gag and pol regions (necessary for viral packaging) by controlling the splicing of the gag/pol messenger RNA via a rev responsive element (RRE).
Another example of the use of the rev protein to control gene expression was described by Yu et al. (J. Virol. 70(7):4530-4537, 1996). They used the expression of tTA to regulate the expression of both HIV-1 rev and envelope proteins which were simultaneously under the regulation of a single TRE containing tet operator. The rev protein then in turn regulates expression of the viral envelope protein, via an RRE, as well as the expression of the gag/pol messenger RNA via another RRE. While transcription of the gag/pol coding sequences was regulated by another promoter, its expression was directly regulated by the rev protein and thus indirectly regulated by tTA.